Biotechnology research, including biological, biomedical, genetic, fermentation, aquaculture, agriculture, forensic and environmental research, demands the ability to identify biological molecules both inside and outside cells. Typically, fluorescing dyes are used to assist in the detection of such biological molecules and in general involve coupling a fluorophore to a probe molecule, having a specific affinity towards a target biological molecule. The resultant fluorescent tagged probe molecule is used in a biological assay to optically identify the presence of the target biological molecule. Such fluorescing dyes, however, are often of marginal use because of low signal to noise ratios, low photostability, and lack of sensitivity. In particular, many fluorescing dyes have broad emission spectra and narrow absorption spectra thereby limiting the number of tests that can be performed in a single assay. Specifically, it is difficult to discriminate the fluorescence associated with a particular dye given the high background and the broad emission spectra of the various dyes in a single test.
Recently, quantum dots (also known as semiconductor nanocrystals) have been used as replacements for the traditional molecular fluorescing dyes because they exhibit unique optical properties. In particular, quantum dots have a continuously selectable wavelength emission, have narrow spectral emissions, and broadband absorption. As a result, they are particularly suitable as dye replacements for a variety of applications including fluorescence-based biological assays.
Such biological assay are typically undertaken in aqueous solutions and it is therefore desirable that the quantum dots be water soluble and be capable of stably coupling to a probe molecule. Attempts at making quantum dots water-soluble have involved coating the quantum dots with a hydrophilic coating. Such attempts, however, have resulted in precipitation of the quantum dots, indicating a lack of tight binding of the hydrophilic coating to the nanocrystal surface. In biological assays that require the quantum dot to couple to a probe molecule, a lack of tight coupling between the probe molecule and the nanocrystal surface will lead to the probe molecule becoming disassociated resulting in inaccurate results of the assay. Attempts at making quantum dots water-soluble and able to stably couple to a probe molecule have also resulted in a marked decrease in the fluorescence quantum yield over time due to oxidation of the nanocrystal. One particular attempt in making quantum dots water soluble involves the use of micelles to solubilize quantum dots and is described in U.S. Pat. No. 6,319,426 to Bawendi. The micelles that are formed using the reagents described Bawendi, however, are not stable in aqueous solutions.
Therefore, recent work has failed to produce a suitable coating that provides for both water solubility and stable coupling to probe molecules because the coatings themselves are not strongly coupled to the nanocrystal surface and tend to detach along with any probe molecule that have been attached to them.